Use of Deep Learning to Evaluate Tumor Microenvironmental Features for Prediction of Colon Cancer Recurrence

Abstract Deep learning may detect biologically important signals embedded in tumor morphologic features that confer distinct prognoses. Tumor morphologic features were quantified to enhance patient risk stratification within DNA mismatch repair (MMR) groups using deep learning. Using a quantitative segmentation algorithm (QuantCRC) that identifies 15 distinct morphologic features, we analyzed 402 resected stage III colon carcinomas [191 deficient (d)-MMR; 189 proficient (p)-MMR] from participants in a phase III trial of FOLFOX-based adjuvant chemotherapy. Results were validated in an independent cohort (176 d-MMR; 1,094 p-MMR). Association of morphologic features with clinicopathologic variables, MMR, KRAS, BRAFV600E, and time-to-recurrence (TTR) was determined. Multivariable Cox proportional hazards models were developed to predict TTR. Tumor morphologic features differed significantly by MMR status. Cancers with p-MMR had more immature desmoplastic stroma. Tumors with d-MMR had increased inflammatory stroma, epithelial tumor-infiltrating lymphocytes (TIL), high-grade histology, mucin, and signet ring cells. Stromal subtype did not differ by BRAFV600E or KRAS status. In p-MMR tumors, multivariable analysis identified tumor-stroma ratio (TSR) as the strongest feature associated with TTR [HRadj 2.02; 95% confidence interval (CI), 1.14–3.57; P = 0.018; 3-year recurrence: 40.2% vs. 20.4%; Q1 vs. Q2–4]. Among d-MMR tumors, extent of inflammatory stroma (continuous HRadj 0.98; 95% CI, 0.96–0.99; P = 0.028; 3-year recurrence: 13.3% vs. 33.4%, Q4 vs. Q1) and N stage were the most robust prognostically. Association of TSR with TTR was independently validated. In conclusion, QuantCRC can quantify morphologic differences within MMR groups in routine tumor sections to determine their relative contributions to patient prognosis, and may elucidate relevant pathophysiologic mechanisms driving prognosis. Significance: A deep learning algorithm can quantify tumor morphologic features that may reflect underlying mechanisms driving prognosis within MMR groups. TSR was the most robust morphologic feature associated with TTR in p-MMR colon cancers. Extent of inflammatory stroma and N stage were the strongest prognostic features in d-MMR tumors. TIL density was not independently prognostic in either MMR group.


Patient Characteristics
The  pathologic features may unlock important quantitative data stored in an H&E slide.QuantCRC was previously trained using 24,157 annotations from 1,054 images from 559 colorectal cancers of all stages (8).Convolutional neural networks were used to segment the image in layers (Fig. 1).Tumor bed is first divided into sections: carcinoma, stroma, TB/PDC, mucin, necrosis, fat, and smooth muscle.Second layer divides stroma into mature (densely collagenous areas with scattered fibroblasts, often with parallel collagen fibers), immature (loose, frequently myxoid stroma with scattered fibroblasts and collagen fibers) and inflammatory (dense clusters of chronic inflammatory cells obscuring stromal cell subtypes).The third layer divides carcinoma into signet ring cell, high-grade, and low-grade.TILs within tumor epithelium were identified at fourth layer.
TB was defined as a single tumor cell or a cluster of <5 tumor cells at the invasive front; clusters of ≥5 tumor cells without gland formation were defined as PDCs (9).

Molecular Analysis
Tumors in the initial study cohort were analyzed for MMR proteins by IHC and if indeterminate, microsatellite instability was tested by PCR.In the vali-dation cohort, MMR status was determined by PCR and/or IHC.Tumors were sequenced to identify a mutation in KRAS (codon 12 or 13) or BRAF c.1799T>A (p.V600E), as described previously (10).

Statistical Analysis
N0147 cohort was used to build recurrence risk prediction models.Univariable Cox regression was used to assess the relationship between morphologic characteristics and time-to-recurrence (TTR; interval between randomization and first disease recurrence).TTR was chosen as the primary outcome variable because recurrence indicates disease progression, is not impacted by treatment after recurrence, and cancer-specific data for death were not available.Tumor morphologic features were analyzed in relationship to TTR using a data-driven approach.For data analyzed as a continuous variable, Kaplan-Meier curves were constructed with data shown by quartile.Data were analyzed as a dichotomous variable when effects were nonlinear, and quartiles were used for the cut-off points.Only features with a univariate P value of <0.05 for association with TTR were included in multivariable models.For morphologic features with a correlation coefficient >0.8 (Spearman rank) with one another, the variable with best P value was included in model selection.Backward selection was used to separately build models in p-MMR and d-MMR tumor groups.
Model covariates were age, sex, performance status, T/N stage, histologic grade, treatment arm, and KRAS, BRAF.Two-sided P values ≤0.05 were regarded as statistically significant (SAS version 9.4) and were not adjusted for multiple comparisons.The performance of the recurrence risk prediction models was further evaluated witihin each MMR group in the validation cohort using the Harrell concordance index (c-index) and AUC, per methods outlined by Royston and Altman (11).This validation is based on predictions from the trained models with estimates that serve to confirm that the variables were clinically relevant.Data collection and analyses were performed by the Alliance Statistics and Data Management Center.

Patient Characteristics
In

Morphologic Features in Relationship to Molecular Variables
Cancers with p-MMR had a higher percentage of immature stroma consistent with desmoplastic reaction in colorectal cancer (Table 1; ref. 12).Among d-MMR tumors, more abundant inflammatory stroma was found which was inversely associated with immature desmoplastic stroma (r = −0.87).Tumors with d-MMR versus p-MMR had higher epithelial TIL densities and increased high-grade histology, mucin, and signet ring cells.Tumors with d-MMR and BRAF VE had higher epithelial TIL density (P-value = 0.01).Among d-MMR tumors, KRAS mutation was associated with increased tumor-stroma ratio (TSR; P-value = 0.040), lower grade (P-value <0.001), and reduced median epithelial TIL density (P-value = 0.003).Tumors with p-MMR and BRAF VE (vs.nonmutated) had increased mature stroma and less necrosis (both P-value ≤0.006).

Validation Cohort
Among p-MMR tumors, the predictive model showed the ability to discriminate by recurrence status (Harrell c-index 0.62; 95% CI, 0.58-0.65)along with 3-year AUC of 0.76.Among patients with d-MMR tumors, the predictive model had lower discrimination ability (c-index 0.57, 95% CI, 0.46-0.68)along with 3-year AUC of 0.67.Among d-MMR tumors, however, the number of recurrences in relation to sample size was lower in the validation compared with the initial cohort.

Discussion
The objective of this study was to examine prognostic value of morphologic features in patients with stage III colon cancer from a phase III clinical trial selected by MMR status with the purpose of developing a recurrence prediction model within each MMR subgroup.We applied a DL algorithm to routine H&E-stained sections of stage III colon carcinomas from a phase III trial of standard FOLFOX-based adjuvant chemotherapy to determine their relative contribution to outcome, and then validated our findings in an external cohort.QuantCRC quantifies 15 distinct tumor morphologic features that had been previously validated against pathologist annotated features in human colorectal cancers (5).Improved quantification of these known pathologic features  may unlock important quantitative data stored in an H&E slide.QuantCRC identified a significant increase in immature desmoplastic stroma in p-MMR compared with d-MMR tumors.In contrast, d-MMR tumors had significantly more inflammatory stroma containing chronic inflammatory cells in addition to higher epithelial TILs and established features of high-grade histology, mucin (13), and signet ring cells (14).Interestingly, d-MMR tumors with mutant KRAS (13.8%) had a significantly higher TSR and lower epithelial TILs.
Among p-MMR tumors in the initial cohort, lower (vs.higher) TSR was found to significantly and independently predict shorter TTR and higher 3-year recurrence rates of 40.2% versus 20.4%.TSR was validated in an external cohort.
In another study where QuantCRC was applied to stage II colon carcinomas, TSR was found to be significantly associated with patient relapse-free survival (5).In human colon cancers, lower TSR (high stroma) was reported to be significantly associated with transcriptomically-determined consensus molecular subtype 4 (CMS4) which exhibits an epithelial-to-mesenchymal transition phenotype (15).The prognostic value of CMS4 was shown to be largely explained by cancer-associated fibroblast (CAF) infiltration score in a multivariable analysis of stage II-III colon cancers (16).As a key component of tumor stroma, CAF functions include collagen deposition and promotion of an immune suppressive tumor microenvironment (TME) which are determinants of progression and metastasis of colorectal cancer (17).Another TME component are TILs which reflect the host antitumor immune response (18,19) and in several reports, TIL density and Immunoscore have been independent prognostic variables in patients with nonmetastatic colorectal cancer (20,21).In our cohort, however, TIL density did not remain in the final multivariable model in either p-MMR nor d-MMR tumors.
Multivariable modeling in d-MMR tumors revealed that the extent of inflammatory stroma, encompassing peritumoral lymphocytic reaction and Crohn's like lymphoid reaction (22,23), was the only morphologic feature that was significantly associated with TTR in the initial cohort.Furthermore, the dichotomized high versus low inflammatory stroma was associated with significantly reduced tumor recurrence at 3 years (13.3% vs. 33.4%).Patients with d-MMR tumors and higher inflammatory stroma showed a trend toward longer TTR in the validation cohort that did not achieve statistical significance, likely due to fewer outcome events compared with the initial cohort (24 vs. 47).
DL may identify biologically important signals embedded in tumor morphologic features which drive distinct prognoses (1)(2)(3).In this regard, QuantCRC can quantify distinct tumor regions and has the potential to be used to guide interrogation using spatial multiomics approaches.Other studies in colorectal cancer using DL and an unsupervised approach (1, 3) were shown to provide prognostic information, yet lacked biological interpretability.Strengths of our study include same stage tumors from a clinical trial cohort with uniform treatment, and a multicenter external cohort used to validate our study results.A limitation is that d-MMR tumors in the validation cohort had fewer outcome events than in the initial cohort which limited statistical power for confirmation of the model.
In conclusion, QuantCRC enables quantitation of distinct tumor morphologic features in whole tumor sections that would not be possible for pathologists to quantify manually (5).Moreover, we demonstrate that this DL algorithm applied to routine colon cancer sections can identify those morphologic features that drive patient prognosis within MMR groups.Furthermore, these features may potentially elucidate pathophysiologic mechanisms driving distinct prognoses within MMR groups.

FIGURE 1
FIGURE 1 QuantCRC detects tumor morphologic features in four layers.TB/PDC, tumor budding/poorly differentiated cluster; TIL, tumor-infiltrating lymphocytes.https://cloud.aiforia.com/Public/MayoUpmcAiforia_Pai/0z9TK9WQComQSW5MOEo_1KieA8U9KX9oCFbc2SQ-ioM0.To see the QuantCRC analysis click on the image, then click on "analyze," and then click on the thumbnail of the image.

TABLE 1
DL-derived tumor morphologic features by DNA MMR status IntroductionDeep learning (DL) can detect and quantify distinct morphologic features of colon cancers that pathologists may be unable to recognize, require diseasespecific expertise, and are time intensive.Among patients with stage III colon cancer, decision-making for adjuvant chemotherapy and its duration (3 vs. 6 months) is determined entirely by T and N stage.Although validated prognostic biomarkers could facilitate adjuvant treatment decisions, very few have been sufficiently validated for clinical application.Importantly, DL may identify biologically important signals embedded in morphologic differences that may underlie tumor subtypes with distinct prognoses (1-3).Other advantages of DL include enhancing pathology interpretive accuracy, increasing efficiency, and reducing interobserver variability (4).To date, however, most DL algorithms have been trained on cancer case series lacking data for DNA mismatch repair (MMR) status which influences pathomorphology and prognosis.
the initial study cohort, 380 of 402 tumors met quality control criteria (d-MMR, n = 191; p-MMR, n = 189).Of these, 183 (48.2%) were female, mean age was 59.4 (SD 12.4) years, and there were 99 recurrences at a median followup of 5 years.BRAF VE was detected in 111 (30.7%) and KRAS mutation in 92 (24.5%) tumors.Patient characteristics stratified by MMR status are shown in Supplementary Table S1.The validation cohort includes 176 tumors with d-MMR and 1,099 with p-MMR.

TABLE 2
Multivariable models for the prediction of TTR by morphologic features and clinicopathological variables in initial cohort [p-MMR (n = 189) or d-MMR (n = 191) stage III colon cancers] a Global likelihood ratio P-value.b Not estimable due to no events in the "T1 or T2" level.

TABLE 3
Multivariable models for the prediction of TTR by morphologic features and clinicopathological variables in the validation cohort [p-MMR (n = 1,094) or d-MMR (n = 176) stage III colon cancers] a Global likelihood ratio P-value.